The arthropod-borne viruses within the Flaviviridae family include West Nile, Japanese encephalitis (JE), St. Louis encephalitis, and tick-borne encephalitis viruses, that are important human pathogens, causing outbreaks of meningoencephalitis in humans in many regions of the world with fatality rates between 10 and 50%. WN and SLE are endemic in the North America, and WN was the major cause of viral encephalitis in the USA. During the 1999-2011 outbreaks, there were at least 31,414 reported human cases of WN illness that resulted in 1263 deaths. No established specific antiviral therapy or a licensed human vaccine is available to date to treat and prevent SLE and WN diseases. Tick-borne encephalitis is a severe neurologic disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBEV during the past 2 decades emphasizes the need for an alternative vaccine that will induce a more durable immunity and protection against TBEV. In an effort to develop the efficacious live attenuated vaccines against neurotropic flaviviruses, we explored several strategies, and progress toward this goal in the past year is reviewed below. In the first approach, the live attenuated TBEV or WN vaccine candidates are being developed in the Neurotropic Flaviviruses Section (NFS) of the LID using a strategy based on chimerization of a neurovirulent TBEV or WN with a non-neuroinvasive dengue-4 flavivirus (DEN4) and introduction of attenuating mutations. The introduction of a 30 nucleotide deletion in the 3' non-coding region (3'NCR) of chimeric TBEV/DEN4 or WN/DEN4 genome that contains the structural protein genes of a highly virulent TBEV or WN virus has a significant attenuating effect on neurovirulence, neuroinvasiveness, and neuropathogenesis in adult mice. However, in a more susceptible animal model such as newborn or immunodeficient mice, TBEV/DEN4d30 exhibited a high level of neurovirulence in the CNS of suckling mice and monkeys compared to that observed for widely used live attenuated yellow fever 17D vaccine virus. Further attenuation of TBEV/DEN4d30 was achieved by introducing mutations, such as amino acid substitutions in the envelope E (Lys315 > Glu) and NS5 (Asp654Arg655 >AlaAla) proteins, that reduced the virus replication in the brain of mice. In the extensive preclinical evaluation in mice, we have identified the TBEV/DEN4d30/E315/NS5654,655 virus as the most promising vaccine candidate against TBEV since it exhibited an acceptable balance between attenuation and immunogenicity. A vaccine candidate is safe, attenuated, able to provide protection against severe TBEV challenge in mice, and has a limited potential for transmission by Aedes mosquitoes and Ixodes ticks. In the future studies, it will be necessary to examine the neurovirulence of a TBEV/DEN4d30/E315/NS5654,655 seed virus in the CNS of non-human primates prior to testing it in the clinical trials in humans. A live attenuated WN vaccine (WN/DEN4d30 virus) developed in the NFS was found to be well-tolerated, safe, and induced a potent and durable WN antibody response in healthy adult volunteers in Phase I clinical trials at the Johns Hopkins School of Hygiene and Public Health. In FY2012, the additional evidence of vaccine safety for the CNS was obtained in comparative studies of neurovirulence and neuropathogenesis in four groups of rhesus monkeys following intrathalamic inoculation with the wild-type WN virus or the WN/DEN4d30, DEN4d30, or YF 17D vaccine. The clinical and virology data indicated that the WN/DEN4d30 vaccine is the most attenuated virus in the CNS compared to either comparator virus as demonstrated by a lack of virus replication in the brain and spinal cord and the absence of clinical signs of neurological disease. Preliminary histopathology data indicated that the mean scores of virus-associated pathology for the entire CNS of WN/DEN4d30- or DEN4d30-infected monkeys were similar or lower than those of YF 17D-infected monkeys. These results suggest that the WN vaccine virus is sufficiently attenuated for the CNS as compared to YF 17D vaccine and support its further large-scale clinical trials in humans, including the risk groups of elderly volunteers. In the second approach for the design of safe and effective live flavivirus vaccines, we explored the ability of the CNS-expressed cellular microRNAs to modulate the neurotropism and pathogenesis of flaviviruses. The inclusion of a single target copy for the brain tissue-expressed miRNAs into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in adult mice. However, we found that the efficacy of miRNA-mediated inhibition of virus replication in the immature CNS of suckling mice depends on the genetic stability of the miRNA-targeted virus and the virus can revert to a virulent phenotype. In FY2012, we demonstrated that multiple miRNA-targeting of flavivirus genome in the 3'NCR strongly attenuated the virus neurovirulence and pathogenesis in the developing mouse CNS. Importantly, virus escape from miRNA-mediated suppression occurs exclusively through the deletion of inserted miRNA-targets and results in the loss of the viral genome sequence located between the two most distant miRNA targets. These findings offer a general strategy to control the virus escape and reversion to a virulent phenotype: a simultaneous miRNA-targeting of the viral genome at many different functionally important regions (5'NCR, ORF, and 3'NCR) could prevent virus escape from miRNA-based attenuation since a deletion of the targeted genomic sequences located between the inserted miRNA-binding sites would be lethal for the virus. Currently, we are exploring the effect of the simultaneous miRNA-targeting of viral genome in the 3'NCR and between the E and NS1 genes; a set of four viruses that contained three targets for brain-expressed mir-124 and/or mir-9 miRNAs between the E and NS1 genes and three targets for mir-124 miRNA in the 3'NCR have been generated and will be tested in the CNS of newborn mice. Thus, our findings indicate that a microRNA-targeting of the viral genome for cellular miRNAs expressed in the CNS is an effective new strategy to control the tissue tropism and pathogenesis of highly virulent neurotropic flaviviruses and could provide a new basis for design of safe and effective live virus vaccines.